1

A secreted peptide acts on BIN2-mediated phosphorylation of ARFs

to potentiate auxin response during lateral root development

Hyunwoo Cho1*, Hojin Ryu1*, Sangchul Rho2, Kristine Hill3,4, Stephanie Smith3,

Dominique Audenaert5,6, Joonghyuk Park1, Soeun Han1, Tom Beeckman5,6, Malcolm

J. Bennett3,4, Daehee Hwang2, Ive De Smet3,5,6 & Ildoo Hwang1

1Department of Life Sciences, Pohang University of Science and Technology, Pohang

790-784, Korea

2School of Interdisciplinary Bioscience and Bioengineering, Pohang University of

Science and Technology, Pohang 790-784, Korea

3Division of Plant and Crop Sciences, School of Biosciences University of

Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, United Kingdom

4Centre for Plant Integrative Biology, University of Nottingham, Sutton Bonington

Campus, Loughborough LE12 5RD, United Kingdom

5Integrative Plant Biology division, Department of Plant Systems Biology, VIB,

Technologiepark 927. 9052 Ghent, Belgium

6Department of Plant Biotechnology and Bioinformatics, Ghent University,

Technologiepark 927, 9052 Ghent, Belgium

CONTACT

Correspondence: ihwang@postech.ac.kr (I. H.)

ADDITIONAL FOOTNOTES

*These authors contributed equally to this work.

2

ABTRACT

The phytohormone auxin is a key developmental signal in plants. To date, only auxin

perception has been described to trigger the release of transcription factors termed

AUXIN RESPONSE FACTORs (ARFs) from their AUXIN/INDOLE-3-ACETIC

ACID (AUX/IAA) repressor proteins. Here, we show that phosphorylation of ARF7

and ARF19 via BRASSINOSTEROID-INSENSITIVE2 (BIN2) can also potentiates

auxin signalling output during lateral root organogenesis. BIN2-mediated

phosphorylation of ARF7 and ARF19 suppresses their interaction with AUX/IAAs,

and subsequently enhances the transcriptional activity to their target genes LATERAL

ORGAN BOUNDARIES-DOMAIN16 (LBD16) and LBD29. In this context, BIN2 is

under the control of the TRACHEARY ELEMENT DIFFERENTIATION

INHIBITORY FACTOR (TDIF) – TDIF RECEPTOR (TDR) module. TDIF-initiated

TDR signalling directly acts on BIN2-mediated ARF phosphorylation, leading to the

regulation of auxin signalling during lateral root development. In summary, this study

delineates a TDIF-TDR-BIN2 signalling cascade controlling auxin perception-

independent regulation of ARF and AUX/IAA interaction during lateral root

development.

3

INTRODUCTION

Spatial and temporal regulation of auxin signalling is essential for diverse

developmental processes in plants1, and the nuclear auxin response mechanism has

been well characterized2,3. In brief, auxin maxima formation through its graded

distribution promotes the interaction between its co-receptors, TRANSPORT

INHIBITOR1 (TIR1)/AUXIN-SIGNALING F-BOX PROTEIN (AFB) F-box

proteins and AUX/IAA transcriptional repressors, which triggers degradation of

AUX/IAAs by E3 ubiquitin-ligase SCFTIR1/AFBs complexes4. At low auxin levels,

AUX/IAA repressors are stable and interact with ARF transcription factors, and

recruit co-repressor complexes leading to suppression of auxin-responsive genes1,5. At

higher auxin levels, degradation of AUX/IAAs allows free ARFs to initiate

transcription of auxin-responsive genes2,3. In Arabidopsis, interactions between 6

TIR1/AFB auxin receptors, 23 ARF proteins, and 29 AUX/IAA repressors are

associated with diverse auxin responses during plant growth and development1.

Distinct expression patterns of these central components, and remarkable differences

in binding affinities between TIR1/AFBs and AUX/IAAs could potentially result in a

broad range of auxin signalling outputs6. While the molecular basis of auxin

perception and subsequent proteolysis of AUX/IAAs has been elucidated, the

regulatory mechanism underlying how ARFs determine auxin signalling output and

dissociate from the repressor complex is still unclear.

One important, well- characterised developmental process that is dependent on the

tight regulation of ARFs and AUX/IAAs is lateral root (LR) initiation, patterning and

emergence7-9. The auxin response module composed of ARF7 and ARF19 along with

the target genes LBD16 and LDB29 controls LR organogenesis via auxin-dependent

4

degradation of AUX/IAAs, such as SOLITARY ROOT (SLR)/IAA14 and

MASSUGU 2 (MSG2)/IAA1910,11.

GLYCOGEN SYNTHASE KINASE3 (GSK3) is a key regulator of diverse

developmental processes in animals12. In Arabidopsis, distinct expression patterns of

GSK3-LIKE genes have been observed in various developing organs13,14. However,

molecular links connecting these genes to development are largely unknown. The

plant GSK3 BIN2/ULTRACURVATA1 (UCU1)/DWARF12 (DWF12) is a critical

regulator in brassinosteroid (BR) signal transduction and stomata development15,16.

BIN2 kinase targets YODA (YDA) and SPEECHLESS (SPCH) in stomata

development15,17, and negatively regulates BR signalling by phosphorylation-

dependent inactivation of two BR-related key transcriptional regulators, bri1 EMS

SUPPRESSOR1 (BES1) and its homolog BRASSINAZOLE RESISTANT1

(BZR1)18-20. The negative action of BIN2 in BR signalling is directly relieved by a

phosphatase BSU121. In addition to its action in BR signalling, BIN2 has been

implicated in the regulation of auxin signalling22,23. A gain-of-function allele of BIN2,

ucu1, showed auxin hypersensitivity in root elongation22, and BIN2 appeared to

attenuate DNA-binding affinity of ARF2, a repressor of auxin signalling23. However,

it is largely unknown how BIN2 action is integrated into auxin signalling pathways

during plant growth and development.

Here, we show that the TDIF-TDR-BIN2 signalling module directly regulates

auxin signalling output during LR organogenesis by controlling the transcriptional

activity of ARF7 and ARF19.

5

RESULTS

BIN2 positively regulates lateral root development

Since BIN2 has been implicated in the regulation of auxin signalling22,23, we

investigated its role in auxin-mediated LR development. We examined the LR

phenotype of gain-of-function BIN2 mutants, dwf12-1D-gof and bin2-1-gof, which

displayed a brassinosteroid (BR)-insensitive short root24,25, and we observed an

increased LR density compared to wild-type (Fig. 1a-c). In contrast, loss-of-function

BIN2 mutants, bin2-3-lof13 and bin2-3 bil1 bil226 displayed reduced LR density

compared to the wild-type (Fig. 1a, b). To eliminate the possibility that the LR

phenotypes of dwf12-1D-gof and bin2-1-gof are due to their relatively short root

length, the BR-insensitive mutant bri1-116 was also examined. Indeed, while bri1-

116 displayed a shorter root phenotype, this was not associated with increased LR

density compared to wild-type (Fig. 1d). Furthermore, the bin2-3 bil1 bil2 mutant

phenotype was recapitulated in seedlings treated with bikinin, a GSK3 specific

inhibitor27 (Fig. 1a, b).

The involvement of BIN2 in root branching was further supported by

examination of the pBIN2-GUS reporter line18. BIN2 expression was detected in

xylem pole pericycle cells, epidermal cells and cortex in the basal meristem, but

restricted to the vasculature in the elongation zone (Fig. 1e and Supplementary Fig.

1a). During LR development, BIN2 expression was observed in LR initiation sites and

in the basal part of the dome-shaped LR primordium (Fig. 1e and Supplementary Fig.

1a).

To evaluate the physiological basis of the BR signalling pathway in LR

development, we examined the role of the BR receptor BRI1, the BR biosynthesis

enzyme DWARF4 (DWF4), and the downstream components BRI1 SUPPRESSOR 1

6

(BSU1), BES1 and BZR1. Interestingly, DWF428 and BRI129 overexpressing plants

exhibited higher density of LR primordia than wild-type, while BSU1

overexpressing30 and gain-of function bes1-D20 and bzr1-1D19 plants did not (Fig. 2a).

Furthermore, BR treatment further increased LR development in bin2-1-gof, but not

in the bri1-116 mutant, and BIN2 overexpression increased LR development in the

loss-of-function bri1-5 mutant (Fig. 2b-d). These results indicate that BIN2 positively

regulates LR initiation, and that BR action in LR development is bifurcated upstream

of BSU1, an inhibitor of BIN2.

BIN2 enhances auxin response during lateral root development via ARF7 and

ARF19

The BIN2 expression patterns during LR development and in the vasculature

overlapped with several well-known auxin-related transcriptional regulators for LR

development, including ARF5, ARF7, ARF19, IAA1, IAA3, IAA12, IAA14, IAA18, and

IAA19 (Supplementary Fig. 1b and c)10,11,31,32. Thus, we investigated if BIN2 is

connected to auxin signalling during LR development. First, we found that dwf12-1D-

gof was hypersensitive to auxin with respect to primary root growth, while bin2-3-lof

was less sensitive compared to wild-type (Fig. 3a). Next, after auxin depletion by

NPA treatment, the auxin-responsive reporter pDR5-GUS was more rapidly and

strongly activated by auxin in bin2-1-gof than in wild-type, particularly in the

pericycle (Fig. 3b and Supplementary Fig. 2a). Moreover, expression of LBD16 and

LBD29, which are marker genes for lateral root initiation that function directly

downstream of ARF7 and ARF19, was higher in bin2-1-gof and lower in bri1-116

compared to wild-type (Supplementary Fig. 2b). To further confirm the in planta

molecular action of BIN2 during auxin-mediated LR development, auxin sensitivity

7

of bin2-1-gof, dwf12-1D-gof and bin2-3 bil1 bil2 loss-of-function mutants was

evaluated with respect to LR formation and expression of LBDs (Supplementary Fig.

2c, d). Compared to wild-type plants, in the presence of 1 μM auxin, dwf12-1D-gof

mutants exhibited a 50% increase in LR density, while bin2-3 bil1 bil2 triple mutants

showed a 20% lower LR root density (Fig. 3c). Compared to wild-type, dwf12-1D-gof

consistently exhibited hypersensitivity to exogenous auxin in a time-dependent

manner, as determined by the expression of LBD16 and LBD29. However, LBD16

and LBD29 expression was reduced in bin2-3 bil1 bil2 mutant in the presence of

exogenous auxin (Supplementary Fig. 2d). Consistently, bikinin treatment reduced

LBD expression and auxin-mediated LR development in a dosage dependent manner

(Supplementary Fig. 2e and Fig. 3d). Taken together, these results suggested that

BIN2 functions to increase auxin-responsive gene expression. Consistent with this

mechanism, co-expression of wild-type BIN2 and the auxin-responsive reporter

pGH3-LUC significantly induced reporter expression in a transient protoplast

system33 in a dose-dependent manner (Fig. 3e and Supplementary Fig. 2f).

Additionally, co-expression of a gain-of-function BIN2E263K (dwf12-1D) activated the

GH3 reporter more strongly than BIN2, and synergistically enhanced the reporter

activity in the presence of auxin (Fig. 3e).

We then examined the ability of other Arabidopsis group II GSK3 proteins to

regulate auxin signalling using the pGH3-LUC reporter system. While BIN2-LIKE1

(BIL1) increased GH3 promoter activity to the same degree as BIN2, BIL2 did not

significantly enhance GH3 expression (Supplementary Fig. 3a). Consistent with a

positive regulatory function for BIL1, the bil1 null mutant showed 10% fewer

emerged LRs than wild-type (Supplementary Fig. 3b, c). As expected, both bil1 bin2-

3 and bin2-3 bil1 bil2 loss-of-function mutants exhibited a similar reduction of

8

approximately 20% in LR primordia density (Supplementary Fig. 3c). Taken together,

these results indicate that BIN2 and BIL1 function redundantly to stimulate auxin

signalling during LR development.

To genetically dissect BIN2 action in auxin-mediated LR formation, bin2-1-

gof was crossed with various auxin signalling defective mutants. For this purpose, the

following alleles were used: arf7-1 and arf19-1 (loss-of-function alleles of ARF7 and

ARF19, respectively10,31), tir1-1 (a loss-of-function allele of the auxin receptor

TIR134), and msg2-1 (a gain-of-function allele of IAA19 encoding an auxin-

insensitive, constitutively stable repressor protein11). The bin2-1- gof mutant slightly

restored the arf7-1 LR phenotype, which was possibly due to the presence of the

functional ARF7 homolog, ARF19 (Fig. 3f and Supplementary Fig. 3d, e). Indeed,

bin2-1-gof failed to rescue the arf7-1 arf19-1 double mutant lateral rootless phenotype

(Fig. 3f). To exclude the possibility that alterations in TIR1-mediated degradation of

AUX/IAAs caused the observed lack of suppression, bin2-1, msg2-1 bin2-1-gof and

tir1-1 bin2-1-gof double mutants were analysed. bin2-1-gof rescued msg2-1 and tir1-1

LR phenotypes, as evidenced by the significant increase in emerged LR density (Fig.

3f, g). These results suggested that BIN2 functions either downstream of TIR1 or in

parallel with TIR1-mediated auxin signalling during LR development. Additionally,

BIN2-mediated activation of LR development required ARF7 and ARF19. This

prompted the hypothesis that BIN2 may relieve ARF7 and ARF19 from their

repressor complex, thus leading to their activation irrespective of TIR1 activity during

LR organogenesis.

BIN2 directly phosphorylates ARF7 and ARF19

9

To elucidate the molecular mechanism of BIN2 action on auxin signal

transduction, we tested whether BIN2 directly interacts with ARF or AUX/IAA

proteins. In an in vitro pull-down assay with GST-BIN2, HA-tagged ARF7 was

pulled down (Fig. 4a). Specifically, the QSL-rich C-terminal domain, including the

middle region and AUX/IAA dimerization domain (d2 ARF7), but not B3 DNA-

binding domain (d1 ARF7), directly interacted with BIN2 (Fig. 4b). In contrast,

AUX/IAA proteins did not interact with BIN2 (Supplementary Fig. 4).

We next determined whether ARF7 was phosphorylated by BIN2. BIN2,

BIN2E263K and BIL1 increased the intensity of high molecular weight bands of ARF7

in a gel shift assay (Fig. 4c). The higher molecular weight band shift of ARF7 was

abolished by alkaline phosphatase (CIAP) treatment (Fig. 4d), indicating that the

ARF7 mobility shift was due to phosphorylation by BIN2. Conversely, a kinase-dead

loss-of-function mutant BIN2K69R and BIL2 failed to increase the intensity of the

phosphorylated ARF7 band (Fig. 4c). In addition, phosphorylated ARF7 was highly

enriched in dwf12-1D-gof plants (Fig. 4e), and BIN2 interacted with ARF19 and

shifted the mobility of ARF19 proteins (Fig. 4f, g). In vitro kinase assays revealed

that BIN2 induced the phosphorylation in the QSL-rich C-terminal domain of ARF7

(Fig. 4h). LC-MS/MS analysis of the phosphorylated QSL-rich C-terminal domain of

ARF7 showed that BIN2-dependent phosphorylation occurred at S698 and S707 (Fig.

4i). Taken together, these results suggest that BIN2 directly interacts with and

phosphorylates ARF7 and ARF19.

BIN2-mediated phosphorylation induces ARF7 and ARF19 activity by

attenuating their interaction with AUX/IAAs

10

Next, the ability of BIN2 to affect auxin signalling by modulating ARF7 and

ARF19 activity via phosphorylation was investigated. BIN2 enhanced pGH3-LUC

activity, and co-expression of BIN2 with either ARF7 or ARF19 further enhanced

reporter expression (Fig. 5a, b). In contrast, the kinase dead BIN2K69R mutant exerted

a dominant negative effect, reducing ARF7-induced activation of the reporter by 60%

(Fig. 5a). These data indicate that BIN2 enhances ARF7 and ARF19-mediated

transcriptional activation.

BIN2-mediated phosphorylation may enhance ARF7 activity by blocking its

interaction with AUX/IAA repressors. Thus, we investigated whether phosphorylation

by BIN2 affects the association of ARF7 with AUX/IAAs. Among the 29 members of

Arabidopsis AUX/IAAs, IAA1, IAA3, IAA14, IAA18, and IAA19 are involved in LR

initiation32. While hypo-phosphorylated ARF7 proteins were precipitated together

with GST-IAA1, GST-IAA3, GST-IAA14, GST-IAA18, and GST-IAA19, hyper-

phosphorylated ARF7 proteins were only weakly pulled down with these AUX/IAA

proteins in the presence of BIN2 (Fig. 5c and Supplementary Fig. 5). However,

BIN2K69R did not affect the association of ARF7 with AUX/IAA proteins (Fig. 5c).

Furthermore, substitution of both S698 and S707 with Ala (ARF7S698A/S707A, ARF7m)

could not interfere with the interaction with AUX/IAA proteins in the absence and

presence of BIN2 (Fig. 5c). We then examined if phosphorylation affects ARF7-

mediated auxin response. ARF7m itself was able to activate pGH3-LUC as much as

wild-type ARF7, and its repressor IAA19 inhibited the activity of the reporter (Fig.

5d). Interestingly, IAA19 suppression of the reporter activity was compromised by

BIN2 together with ARF7, but not with ARF7m. Consistently, overexpression of

ARF7 in arf7-1 (referred to as ARF7 arf7-1) restored the LR defects of arf7-1 to wild–

type levels, but ARF7m overexpression in three independent ARF7 transgenic plants

11

with similar protein levels (referred to as ARF7m arf7-1) only partially recovered the

LR defects of arf7-1 (Fig. 5e). Furthermore, bin2-1-gof increased the LR formation of

ARF7 arf7-1 transgenic plants, but not ARF7m arf7-1 plants (Fig. 5f). We therefore

concluded that BIN2-mediated phosphorylation of ARF7 attenuates the interaction of

ARF7 with AUX/IAAs and leads to the activation of auxin signalling. The ability of

BIN2-mediated phosphorylation to alter the interaction between ARF7 and IAA19

was further supported by the rescue of msg2-1 by bin2-1-gof (Fig. 3g).

We next investigated whether AUX/IAAs dissociated from phosphorylated

ARFs could be more rapidly degraded via the TIR1 pathway. To test this possibility,

the effect of ARF7 phosphorylation on the relative amount of interacting proteins in

TIR1-ARF7-IAA19 complexes was tested. Co-expression of BIN2 attenuated the

interaction of IAA19 with ARF7, and BIN2 increased the interaction of IAA19 with

TIR1 (Fig. 6a and Supplementary Fig. 6a). We then examined the protein level of

IAA19 in wild-type or bin2-1-gof plants. The proteolysis of IAA19 proteins occurred

more rapidly in bin2-1-gof than in wild-type, but bikinin treatment effectively

inhibited the IAA19 protein degradation (Fig. 6b). Consistently, the fluorescence of

DII-VENUS, which has no dimerization domain with ARFs, was not significantly

affected by bikinin treatment (Supplementary Fig. 6b). These results suggested that

BIN2-mediated phosphorylation of ARF7 diminishes its interaction with AUX/IAA

proteins and subsequently enlarges the pool of free ARF7 proteins by facilitating

degradation of their repressor AUX/IAAs.

We next examined if BIN2-mediated phosphorylation of ARF7 and ARF19

affects their binding to target promoters using a chromatin immunoprecipitation assay.

While DNA binding of ARF7 and ARF19 to the promoters of LBD16 and LBD29 was

increased in the presence of auxin, bikinin treatment suppressed the auxin-induced

12

enhancement of DNA binding (Fig. 6c, e). Furthermore, the dwf12-1D-gof enhanced

DNA binding of ARF7 to their target promoters (Fig. 6d). The bin2-1-gof further

increased auxin-induced enhancement of ARF19 binding, but bikinin treatment

compromised the enhancement of ARF19 binding in bin2-1-gof, to the LBD29

promoter (Fig. 6f). Together, these results indicated that phosphorylation of ARFs

could increase the pool of active ARFs by attenuating the interaction with AUX/IAAs

and enhance their DNA binding capacity.

TDIF–TDR signalling module affects BIN2 during lateral root development

Our results suggested that BIN2 action is directly linked to ARF transcription

factors, but the limited role of BR-mediated regulation of BIN2 in LR development

suggested that other regulatory components could impact on BIN2 activity during this

process. To identify other upstream regulators of BIN2-mediated LR development,

we screened for BIN2 interacting proteins using a yeast two-hybrid system and

identified TDR as a potential interacting partner. TDR is known to function as a

receptor of TDIF (CLE41 and CLE44) peptide in vascular bundle development35.

Yeast two-hybrid assays showed that BIN2 interacts with the kinase domain of TDR

(TDR kd) (Fig. 7a). Furthermore, we could pull down full-length TDR proteins with

GST-tagged BIN2 (Fig. 7b). This interaction suggested that TDR might be involved

in LR development via the regulation of BIN2. To test this possibility, we first

examined the expression pattern of pTDR-GUS in roots. TDR was expressed in the

vasculature, in pericycle cells and during LR initiation, which overlapped with BIN2

expression (Fig. 1e and Fig. 7c). Consistently, a loss-of-function tdr mutant displayed

altered LR density compared to wild-type (Fig. 7d).

13

We then investigated whether the TDIF–TDR signalling module plays a role

in BIN2-mediated LR development. The LR density of wild-type plants increased

following TDIF treatment in a dosage dependent manner (Fig. 7d). However,

inhibition of BIN2 activity by bikinin treatment or a loss-of-function tdr mutation

completely suppressed the positive effect of TDIF on LR development (Fig. 7d).

TDIF treatment increased the phosphorylation of ARF7 in plants, and its functional

receptor, TDR, was required for the phosphorylation. However, bikinin completely

blocked TDIF-mediated phosphorylation of ARF7 (Fig. 7e). Furthermore, TDIF only

increased the LR density of arf7-1 mutants expressing wild-type ARF7, but not

mARF7 (Fig. 7f). Exogenous TDIF further increased LR formation in bin2-1-gof

compared to wild-type plants, but bin2-3-lof rarely responded to TDIF (Fig. 7g).

These results implied that TDIF–TDR signalling could affect the transcriptional

activity of ARF7 by BIN2-mediated phosphorylation. To examine this idea, we

determined the effects of TDIF–TDR signalling module on ARF7-mediated activation

of pGH3-LUC. Interestingly, exogenous TDIF or expression of TDR itself did not

affect the pGH3-LUC activity, but TDIF treatment significantly promoted the reporter

activity in the presence of TDR (Fig. 7h). Furthermore, ARF7-mediated reporter

activation was enhanced by BIN2, and co-expression of TDR with TDIF treatment

further increased BIN2-mediated activation of ARF7 activity. These data indicated

that the TDIF–TDR module affects BIN2-mediated auxin signalling output during LR

organogenesis.

DISCUSSION

Auxin is involved in nearly every aspect of plant development through a

relatively simple signalling circuit in which de-repression of ARFs following auxin

14

perception activates a transcriptional response1. However, it has been uncertain how

auxin could provide a wide range of signalling outputs for plant growth and

development. For example, several combinations of auxin co-receptor machineries,

especially between TIR1/AFB receptors and AUX/IAA repressors, could modulate

auxin sensitivity6. Our study revealed another regulatory module in auxin signalling

that involves phosphorylation-dependent regulation of transcriptional activity of

ARFs during LR development via the TDIF–TDR–BIN2 signalling cascade.

BIN2 associated with the TDIF–TDR module positively regulates auxin-

mediated LR development by phosphorylating ARF7 and ARF19. This alleviates the

interaction between these ARFs and AUX/IAAs, facilitates SCFTIR1-mediated

degradation of AUX/IAAs, and enhances eventually transcriptional activity of ARFs,

possibly by preventing the reformation of an ARF–AUX/IAA complex and

consequently increasing the pool of free ARF proteins. While BIN2 phosphorylates

ARF2 in vitro23 and ARF7 in vivo (Fig. 4e), the two identified phosphorylation

residues of ARF7 are not conserved in ARF2 and other ARF transcriptional activators

although ARF proteins harbour many putative GSK3 phosphorylation sites.

Interestingly, BIN2-mediated phosphorylation of ARF7 and ARF19 enhances their

DNA binding capacity (Fig. 6), while – in contrast – ARF2 phosphorylation reduces

its DNA binding and repressor activity23. The regulation of ARF DNA binding by its

phosphorylation is likely to be a critical regulatory mechanism for diversity and

specificity of auxin signaling during plant growth and development.

BR and auxin interact synergistically during various physiological responses36.

However, this study revealed that BIN2, a negative regulator in BR signalling,

positively regulates auxin signalling during LR development. BRI1, a major BR

receptor, is involved in BR-mediated activation of LR development, but the canonical

15

BR signalling output determined by BSU1, a BIN2 repressor, and downstream

components BES1 and BZR1 are likely not involved in LR development (Fig. 2). It is

plausible that a not-yet identified signalling pathway bifurcated from BRI1-mediated

BR signalling enhances auxin transport, leading to stimulation of LR development37.

Furthermore, BR did not affect BIN2-induced phosphorylation or transcriptional

activity of ARF7, but a GSK3 inhibitor suppressed BIN2-mediated activation of auxin

signalling (Supplementary Fig. 7). These data demonstrate that BR likely plays a

minor role, if any, in the regulation of BIN2 activity during LR development.

Importantly, the group II GSK3 proteins appear to have a functional redundancy in

BR signalling pathways26, but only BIN2 and BIL1 showed positive roles in ARF7

and ARF19-mediated LR development (Supplementary Fig. 3), which suggests

specificity of GSK3 proteins in diverse developmental processes.

In Arabidopsis, cell-cell regulation via small secreted peptides and their

receptors is essential for various developmental processes38. For example, TDIF–TDR

controls xylem differentiation and proliferation of procambium for vascular bundle

development35, and TDIF and its related peptide, CLE42, are also involved in axillary

meristem formation and bud outgrowth39. Here, we showed that the TDIF–TDR

signalling module also regulates LR development via BIN2-mediated control of auxin

signalling. Interestingly, TDIF is mainly expressed in the phloem and its expression is

activated by auxin40. This expression pattern suggests that auxin-induced TDIF

peptides in the phloem move to the pericycle and stimulate the TDR–BIN2 module,

reinforcing auxin signalling for positioning and development of LRs.

Taken together, these events triggered by TDIF–TDR–BIN2 signalling cues

could increase the pool of active ARF7 and ARF19, in harmony with auxin-induced

degradation of IAA proteins, strengthening the signalling output of auxin during LR

16

organogenesis (Fig 7i). This peptide-initiated phosphorylation-mediated control of

transcriptional activity of ARFs provides a broad spectrum of auxin signalling outputs

to specific cells with a graded distribution of auxin during various development

processes. Our results also suggest that a TDIF TDR GSK3-mediated regulatory

module of ARFs is an important node for connecting diverse signalling networks in

auxin-driven plant growth and development.

Full Methods and any associated references are available in the online version of the

paper at http://www.nature.com/naturecellbiology/.

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Supplementary Information is linked to the online version of the paper at

www.nature.com/naturecellbiology.

Acknowledgements

The authors thank D. Weijers for critical reading of the manuscript and useful

suggestions. This work was supported by grants from the Advanced Biomass R&D

Center (2010-0029720) and the National Research Foundation (20110000212) funded

21

by the Korean Ministry of Education, Science and Technology. I.H. and H.R. were

supported by grants from the Next-Generation BioGreen 21 Program (PJ009072,

PJ009516). H.C. and S.H. were recipients of a Brain Korea 21 fellowship. I.D.S. was

supported by a BBSRC David Phillips Fellowship (BB_BB/H022457/1) and a Marie

Curie European Reintegration Grant (PERG06-GA-2009-256354).

Author Contributions

H.C., H.R., S.H., J.P., K.H. and S.S. performed experiments. H.C., H.R. and I.D.S.

and I.H. designed experiments and analysed the data. D.A. analysed gene expression.

S.R. and D.H. performed LC-MS/MS analysis. H.C., H.R., T.B., M.J.B., I.D.S. and

I.H. wrote the manuscript.

Author information

Reprints and permissions information is available at www.nature.com/reprints. The

authors declare no competing financial interests. Readers are welcome to comment on

the online version of this article at www.nature.com/nature. Correspondence and

requests for materials should be addressed to I.H. (ihwang@postech.ac.kr).

FIGURE LEGENDS

Figure 1. BIN2 positively regulates lateral root development. (a, b) BIN2 kinase

activity is essential for proper LR development. Representative twelve-day-old WT

(Ws-2), dwf12-1D-gof [heterozygote (-/+) and homozygote (+/+)], and bin2-3-lof,

bin2-3 bil1 bil2 mutant and bikinin-treated seedlings are displayed (a). LR

primordium (LRP) densities. E, emerged LRs; NE, non-emerged LRP (n=15 plants)

(b). (c) LRP densities of 12-day-old seedlings of WT (Col-0), bin2-1-gof heterozygote

22

(-/+) and homozygote (+/+) mutants (n=12 plants). (d) The LR development

phenotype of 12-day-old seedlings of WT (Col-0), bri1-116, and bin2-1-gof (left-

panel). The primary root lengths and densities of emerged LRs in WT (Col-0), bri1-

116, and bin2-1-gof (+/+) (right panel) (n=12 plants). (e) pBIN2-GUS expression

limited to LRP and vascular tissues at stage I and II (left panel, black arrowheads

indicate dividing pericycle cells) and transverse sections of pBIN2-GUS seedlings

through the LRP (right panel, black arrows indicate xylem pole pericycle cells). e,

epidermis; c, cortex; end, endodermis; p, pericycle. Scale bars, 50 μm. Asterisks

indicate statistically significant differences as compared with wild-type at p<0.05 (*)

and p<0.01 (**). Error bars indicate SEM. Details on the statistical analysis are found

in Methods.

Figure 2. BR plays a minor role in BIN2-mediated lateral root development. (a)

LRP densities of 12-day-old seedlings of WT (Col-0), bri1-116, BRI1 OX, DWF4 OX,

BSU1 OX, bes1-D (pBES1-bes1-D-HA) and bzr1-D mutants (n=15 plants). (b)

Different BL responses of bri1-116 and bin2-1-gof (+/+) in LR development.

Treatment: BL for 5 days (n=20 plants). (c) The LR development phenotype of

twelve-day-old seedlings of WT (Ws-2), bri1-5, and bri1-5 BIN2 OX. (d) LRP

densities of 12-day-old seedlings of WT (Ws-2), bri1-5, and bri1-5 BIN2 OX. BIN2-

HA proteins in the transgenic plants were presented (n=12 plants) (lower). Error bars

indicate SEM (* for p<0.05, ** for p<0.01 by student’s t-test). Details on the

statistical analysis are found in Methods.

Figure 3. BIN2-mediated signalling is integrated into ARF7/19 during lateral

root development. (a) Different auxin responses of bin2 mutants in root elongation.

23

Relative root length of the 12-day-old dwf12-1D-gof [heterozygote (-/+) and

homozygote (+/+)] and bin2-3-lof compared to wild-type. Treatment: IAA for 5 days

(n=12 plants). (b) The bin2-1-gof mutation increases auxin sensitivity in pDR5-GUS

activation. The indicated plants were grown for 72 h in the presence of 10 μM NPA

and then transferred to 1 μM NAA for 1, 3, and 5 h. The arrowheads indicate dividing

pericycle cells. Scale bars, 50 μm. (c) BIN2 increases auxin sensitivity in LR

formation. The densities of emerged LRs in 11-day-old seedlings of WT (Ws-2),

dwf12-1D-gof homozygote (+/+), and bin2-3 bil1 bil2 mutant. Treatment: IAA for 4

days (n=12 plants). (d) Bikinin treatment inhibits auxin-mediated LR development.

The densities of emerged LRs were measured in the presence of indicated

concentration of auxin and bikinin. Error bars indicate SEM (n=15 plants). (e) BIN2

and auxin synergistically enhance the activity of the GH3 promoter. Protoplasts were

co-transfected with pUBQ10-GUS (internal control), pGH3-LUC and the indicated

effector constructs, and treated with 1 μM IAA for 3 h. BIN2 protein levels were

detected using anti-HA antibody (n=4, 20,000 cells per n=1). Full scan image of

western blot is shown in Supplementary Fig. 8. Statistics source data is found in

Supplementary Table 2. (f) bin2-1-gof homozygote mutation suppresses the LR

defects of tir1-1, but not arf7-1, and arf7-1 arf19-1. The densities of the emerged LRs

of 12-day-old seedlings of indicated genotypes are presented (n=16 plants). (g) bin2-

1-gof partially suppresses the defective LR phenotype of msg2-1. The densities of the

emerged LRs of 12-day-old seedlings of indicated genotypes are presented (n=15

plants). Error bars indicate SEM (* for p<0.05, ** for p<0.01 by student’s t-test).

Details on the statistical analysis are found in Methods.

24

Figure 4. BIN2 directly phosphorylates ARF7. (a) BIN2 interacts with ARF7.

Protoplast lysates overexpressing ARF7-HA were incubated with purified GST-BIN2

proteins and pulled down with glutathione sepharose 4B. GST-BIN2-bound proteins

were visualized with anti-HA HRP antibody. An asterisk indicates GST-BIN2

proteins. (b) BIN2 interacts with QSL-rich C-terminal domain of ARF7 (d2 ARF7).

Protoplast lysates overexpressing HA-tagged B3 DNA-binding domain of ARF7 (d1

ARF7) or QSL-rich C-terminal domain (d2 ARF7) were incubated with GST-BIN2 or

GST proteins and pulled down with glutathione sepharose 4B. Precipitated proteins

were visualized by anti-HA antibody. (c) BIN2 kinase activity is required for ARF7

phosphorylation. HA-tagged ARF7 was co-transfected into protoplasts with BIN2-HA,

BIN2E263K-HA, BIN2K69R–HA, or BIL1-HA, BIL2-HA. After 6 h of transfection, the

indicated proteins were visualized with anti-HA antibody. (d) The BIN2-induced

mobility shift was abolished by phosphatase (CIAP) treatment. (e) ARF7 is

phosphorylated by BIN2 in vivo. The phosphorylation status of ARF7 proteins in WT,

dwf12-1D-gof plants harboring 35S-ARF7-HA in the presence or absence of 5 μM

bikinin. The BIN2-induced mobility shift was abolished by phosphatase (CIAP) or

bikinin treatment. (f) BIN2 interacts with ARF19 in vitro. Protoplast lysates

overexpressing ARF19-HA were incubated with GST-BIN2 proteins and pulled down

with glutathione sepharose 4B. GST-BIN2-bound proteins were visualized with anti-

HA HRP antibody. An asterisk indicates GST-BIN2 proteins. (g) BIN2

phosphorylates ARF19 proteins. ARF19-HA was cotransfected into protoplasts with

BIN2-HA. ARF19 and BIN2 proteins were visualized with anti-HA antibody. (h)

BIN2 directly phosphorylates ARF7. GST-tagged BIN2 and the QSL-rich C-terminal

domain of ARF7 (d2 ARF7) were used in an in vitro kinase assay. The autoradiogram

shows protein phosphorylation (upper panel). The protein levels were determined by

25

Coomassie blue staining (lower panel). (i) MS/MS spectra for two major

phosphopeptides of GST-d2 ARF7 showing in vitro BIN2 phosphorylation sites of

ARF7 at serine 698 (left panel) and serine 707 (right panel). GST-d2 ARF7 was

incubated with GST-BIN2 and ATP for 12 h. The proteins were digested by trypsin

and analyzed by LC-MS/MS. Full scan images of western blots are shown in

Supplementary Fig. 8.

Figure 5. BIN2 attenuates the interaction of ARF7 with AUX/IAAs and enhances

its transcriptional activity, leading to activation of auxin response. (a) BIN2

enhances the transcriptional activity of ARF7. The relative fold induction of

luciferase activity was determined (n=4, 20,000 cells per n=1). Protein expression

levels were presented (lower). (b) BIN2 enhances the transcriptional activity of

ARF19. The relative fold induction of luciferase activity was determined (n=4, 20,000

cells per n=1). (c) BIN2-mediated phosphorylation of ARF7 represses its interaction

with IAA19, but mutation of two phosphorylation sites of ARF7 (ARF7m:

ARF7S698A/S707A) blocks BIN2-mediated dissociation with IAA19. (d) BIN2 could not

enhance the transcriptional activity of ARF7m in the presence of IAA19 in

protoplasts (n=4, 20,000 cells per n=1). ARF7, IAA19 and BIN2 protein levels were

detected using anti-HA antibody. (e) The LR defects of arf7-1 are completely rescued

by overexpression of ARF7, but partially by ARF7m (n=25 plants). ARF7-HA and

ARF7m-HA proteins in the transgenic plants were presented (lower). (f) The bin2-1-

gof mutation enhanced ARF7-mediated LR formation, but not ARF7m. The densities

of LRs of WT, arf7-1 35S-ARF7-HA (bin2-1-gof) and arf7-1 35S-ARF7m-HA (bin2-

1-gof) are presented (n=42 plants). Error bars indicate SEM (* for p<0.05, ** for

p<0.01 by student’s t-test). Full scan images of western blots are shown in

26

Supplementary Fig. 8. Details on the statistical analysis are found in Methods.

Figure 6. BIN2 increases DNA binding capacity of ARF7 and ARF19. (a) BIN2-

mediated phosphorylation of ARF7 results in increased binding of IAA19 with TIR1.

Asterisks indicate GST-IAA19 proteins. The levels of GST-IAA19 pull-downed

proteins were normalized to those of the input proteins. The normalized intensities of

the pull-downed proteins in the absence of BIN2 were set to 1, and the relative protein

intensities were presented under the corresponding protein bands (left panel). The

average values of the relative band intensities from three independent experiments

were plotted (right panel). (b) IAA19 proteins are rapidly degraded in bin2-1-gof

plants but stabilized by bikinin. WT (Col-0) and bin2-1-gof homozygote plants

expressing 35S-IAA19-HA were grown in the presence of 5 μM bikinin or mock

control, and incubated for the indicated times with 100 μM of cycloheximide. The

levels of IAA19 proteins were determined with anti-HA antibody (upper panel). The

average value of IAA19 band intensities and half-life of IAA19 proteins from three

independent experiments was calculated (lower panel). Error bars indicate SEM (n=3,

10 plants per n=1). Statistics source data is found in Supplementary Table 2. (c-f)

BIN2 increases the binding of ARFs to the LBD16 and LBD29 promoters. A BIN2

inhibitor, bikinin, represses the binding of ARF7 and ARF19 to the LBD16 and

LBD29 promoters (c, e). The dwf12-1D-gof and bin2-1-gof mutations enhance the

DNA binding of ARF7 and ARF19 to the LBD16 and LBD29 promoter (d, f). The

DNA binding of ARF7 and ARF19 was determined by quantitative RT-PCR. Error

bars indicate SEM (n=6 for ARF7, n=4 for ARF19, 200 plants per n=1). The LBD29

promoters bound to ARF19 proteins in bin2-1-gof were determined with/without 50

μM of bikinin in the presence of 1 μM of 2.4D. The source data is found in

27

Supplementary Table 2. Full scan images of western blots are shown in

Supplementary Fig. 8.

Figure 7. TDIF-TDR module is directly linked to BIN2-mediated auxin signal

activation in lateral root development. (a) BIN2 interacts with the TDR-kinase

domain (TDR kd) in a yeast two-hybrid assay. (b) BIN2 interacts with TDR.

Protoplast lysates overexpressing TDR-HA were incubated with purified GST-BIN2

proteins and pulled down with glutathione sepharose 4B. An asterisk indicates GST-

BIN2 proteins. (c) pTDR-GUS expression limited to LRP and vascular tissues at stage

I and II (upper panel, black arrowheads indicate dividing pericycle cells) and

transverse sections of pTDR-GUS seedlings through the LRP (lower panel, black

arrows indicate xylem pole pericycle cells). e, epidermis; c, cortex; end, endodermis;

p, pericycle. Scale bars, 50 μm. (d) TDR is positively involved in LR development

via GSK3 dependent manner. The densities of emerged LRs of seedlings treated with

indicated concentrations of TDIF with/without 5 μM of bikinin (n=15 plants). (e)

TDIF induces the phosphorylation of ARF7. 35S-ARF7-HA transgenic plants were

treated with 1 μM of TDIF in the absence or presence of 5 μM bikinin (upper panel).

HA-tagged ARF7 was co-transfected into protoplasts with or without TDR-MYC.

After 5 h of transfection, the protoplasts were further incubated with 1 μM of TDIF

for 1 h (lower panel). (f) TDIF increases LR development by BIN2-mediated

phosphorylation of ARF7. The densities of the emerged LRs of indicated genotypes

are presented (n=15 plants). (g) The densities of emerged LRs of WT (Ws-2), dwf12-

1D-gof (+/+), and bin2-3-lof treated with indicated concentrations of TDIF (n=15

plants). (h) TDIF-TDR-BIN2 cascade enhances the transcriptional activity of ARF7

in protoplasts (n=4, 20,000 cells per n=1). ARF7, BIN2, and TDR protein levels were

28

detected using anti-HA or anti-MYC antibody. Error bars indicate SEM (* for p<0.05,

** for p<0.01 by student’s t-test). (i) A model for the TDIF-TDR-BIN2-mediated

regulatory module of auxin signal transduction. Full scan images of western blots are

shown in Supplementary Fig. 8. Details on the statistical analysis are found in

Methods.

a

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0

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4

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WT

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erge

d la

tera

l roo

ts (#

/ cm

) e

0

2

1

3

WT arf7-1

ARF7 ARF7m

#1 #2 #3ARF7Actin

*****

f

**

Em

erge

d la

tera

l roo

ts (#

/ cm

)

ARF7 ARF7m

arf7-1 arf7-1 bin2-1

0

1

2

3

4

d

Rel

ativ

e ac

tivity

0

12

18ARF7 ARF7m

- ++- - +- -

++- +- -

(pG

H3-

LUC

/ p3

5S-R

enilla

)

BIN2IAA19

6

Con

BIN2IAA19ARF7

**

GST-IAA19

GST

ARF7 / GST-IAA19

ARF7 /GST

BIN2

ARF7

ARF7

Input

Input

Con

BIN

2B

IN2K

69R

Con

BIN

2B

IN2K

69R

ARF7m / GST-IAA19

Con

BIN

2

Pulldown

c

0

2

4

6

8

- +- - ++

BIN2ARF19R

elat

ive

activ

ity(p

GH

3-LU

C /

pUBQ

10-G

US

)**

****

b

Rel

ativ

e ac

tivity

(pG

H3-

LUC

/ pU

BQ10

-GU

S)

a

-+- +- -

-+- +- - BIN2K69R

BIN2--- +++ ARF7

02468

10

BIN2ARF7

12 **

***

**

**

a

Input

InputPulldown

ARF7

TIR1

TIR1

GST-IAA19

GST

Pulldown ARF7

+ +-- BIN2GST

GST-IAA19

* *

1.0 0.13

1.0 5.1

GSTGST-IA

A19

ARF7

TIR1

1.2

0.8

0.4

0

6

4

2

0

8

Pulle

d-do

wn

prot

eins

Pulle

d-do

wn

prot

eins

+ BIN2- BIN2

bWT bin2-1

0 30 60 90 (min)IAA19 Actin

1.5 1.9 1.31.0 0.1 0.11.0 0.7 0.41.0 1.30.1

0 30 60 90

bikinin

0 30 60 90

0

0.5

1

1.5

30 60 90 0

Rel

ativ

e IA

A19

prot

ein

leve

l

WTbin2-1bikinin

t1/2=115.7±11.3

(min)

t1/2=81.6±13.5t1/2=155.5±22.9

Rel

ativ

e C

hIP

enric

hmen

t

ARF19ARF19 / bin2-1

LBD290

3

9

6

f

ARF19 / bin2-1+bikinin

3

2

1

0

LBD16 LBD29Rel

ativ

e C

hIP

enric

hmen

t auxin auxin + bikinine

0

4

12

8

ARF19

30

20

10

0

d

Rel

ativ

e C

hIP

enric

hmen

t

ARF7 ARF7 / dwf12-1D

LBD16 LBD29

c

Rel

ativ

e C

hIP

enric

hmen

t

auxin auxin + bikinin

LBD16 LBD29

ARF7

a

GAD /GBK

GBK-BIN2

GAD /

GAD-TDR kd / GBK

GAD-TDR kd / GBK-BIN2

+HIS -HIS

+3 mM AT

i

PARF7/19

ARF7/19

+Auxin

TIR1

AUX/IAA

TIR1

AUX/IAA

TIR1

AUX/IAA

P

Auxin response in lateral root organogenesis

ARF7/19 ARF7/19

ARF7/19

+Auxin

TDR

TDIF

BIN2

TIR1

AUX/IAA

Rel

ativ

e ac

tivity

h

(pG

H3-

LUC

/ p3

5S-R

enill

a)

- +- - ++ + ARF7BIN2TDRTDIF

-- -- - ++ +-- -+ + +- +-+ -- + +- --

0

10

40

50

20

30

ARF7BIN2TDR

****

******

*

g

Em

erge

d la

tera

l roo

ts (#

/ cm

)

0

2

4

6

0 0.5 1

dwf12-1D bin2-3

WT

TDIF (μM)

f

Em

erge

d la

tera

l roo

ts (#

/ cm

) ARF7 / arf7-1ARF7m / arf7-1

WT

0

1

2

3

TDIF (μM)0 0.5 1

*****

e- + + - + -

ARF7

TDIF

ARF7-P

- +

ARF7

TDR

TDIF

bikinin

+

d

Em

erge

d la

tera

l roo

ts (#

/ cm

)

0

1

2

0 0.1 0.5TDIF (μM)

1

3

WTWT+bikinin

tdrtdr+bikinin

4

*** **

cpTDR-GUSStage I Stage II

p p

p

e

endc

p

e

endc

b

TDR

TDR

GST-BIN2

GST

*

Input

TDR/GST

TDR/GST-BIN2

Pulldown